ATCC Mock 20 Strains DNA metabarcoding analysis through PacBio sequencing
The 27F/1492R universal primer sets (Callahan et al., 2019) were used to amplify the full length 16S rRNA gene, all tailed with sample-specific PacBio barcode sequences for multiplexed sequencing. Amplifications were performed using a 25 l mixture containing 2 ng of DNA, 1X Buffer HF, 0.2 mM dNTPs, 0.375 M of each primer (Fwd-Rev), and 1U/L of Phusion High-Fidelity DNA Polymerase. The cycling parameters for amplification of the target gene were standardized as follows: initial denaturation at 98C for 30 seconds, followed by 10 cycles of denaturation at 98C for 10 seconds, annealing at 58C for 30 seconds, extension at 72C for 1 minute, and subsequently 15 cycles of denaturation at 98C for 10 seconds, annealing at 62C for 30 seconds, extension at 72C for 1 minute, with a final extension step of 7 min at 72C. The PCR products (~1.5 kb long) were quality checked on agarose gel and quantified using Qubit 1X dsDNA HS Assay (Thermo Fisher Scientific, Inc.). Each barcoded amplicon was pooled at equimolar concentrations. Then, the SMRTbell Library Construction was performed according to the manufacturer's instructions of the Amplification of Full-Length 16S Gene with Barcoded Primers for multiplexed SMRTbell Library Preparation and Sequencing, using the SMRTbell Express Template Prep Kit 2.0 and the Binding Kit 2.1 (Pacific Biosciences). The SMRTBell library was sequenced on the PacBio Sequel System II platform.